I have neuroscreen-1 (NS-1) cells (theses cell are sub-clones from PC12 cells) which I want to co-transfect with a specific miRNA as well as that miRNAs predicted mRNA target, to see if the miRNA targets that mRNA directly by doing luciferase assays. Since this experiment is based on miRNA seq results from adult rat left dorsal hippocampus brain samples, I want to know if I should differentiate these cells before conducting this experiment.
Why don't you use 293T cells to do the reporter assay for miRNA-target validation?
These hek cells r always good guys at reporter assay always best result from them!
well, PC12 differentiated or not are not exactly hippocampal neurons, so why dont you just start with the cells are they are to validate the miRNA? It is hard enough to get what you want into differentiated cells, particularly for bulk assays. In fact nowadays inducible system are working better and you might consider just introducing your miRNA in inducible form in your cells, then differentiate and then test, it is a bit of work at first but it is really easier once your cells are ready.
Thanks guys. Initially we weren't sure what kinds of experiments we were going to do and then we were looking for a neuronal cell line and saw the NS-1 cells and all the advantages of using these cells, so that's why we bought them. Also, I saw another article that also did functional miRNA work and they used this cell line and differentiated with NGF-B. In essence if the correct agents are present (such as DICER & all the other enzymes that are involved in the processing of miRNAs) then if the miRNA does target the mRNA it should work in NS-1 cells, right, since I co-transfect with them, or am I misunderstanding?
It should work, but the cell line is not really a neuron (neuronal-like may be) being originally from adrenal gland cancer, so I am not following why you us them at all.
How is the differentiation figure in your project if you transfect with both miRNA and target? If it does not, there is no reason to bother about differentiation since it makes cells harder to transfect by classical method, in addition it requires time. In fact if I understand correctly what you want to do, I would be again more interested in using a cell line that transfects well, like HEK, CHO, etc.. in addition I would make sure the cell line will not be the type that might express miRNA which could silence my target mRNA (so choosing a different specie is also a good idea) thus introducing false positive results.
Hi Stephanie,
It should work in NS1/PC3cells. No need for the differentiation if you interested in reporter only. I would say that you can use them if you need rat cells. In the proposed experiment format (miRNA+reporter) you can probably use any cells that are transfected well.
I agree with Genevieve Bart and Wen- Yuan Hu , choose a cell line where it is easier to perform the reporter assay experiment like 293T,
Talking about differentiating cells do you have any suggestions about this problem ? I am trying to measure by qrtPCR the level of miRNAs and their putative targets during differentiation of two different cell lines , what is the best way to compare them ? thanks Marcella
Thanks Marcella. There are two publications you can have a look at that did something similar to what you want to do. These are Vreugdenhil et al., 2009 in Endocrinology 150: 2220-2228 and Dijkmans et al., 2008 in Journal of Neurochemistry 105: 2388 - 2403
Since your assay is between transfected miRNA expressing plasmid and reporter luciferase assays, therefor it is not really important which cell you use Hek293 are easy to transfect that is way everyone use them. But you have to show that when you transfect a mimic miRNA to you NS-1 cells the endogenic putative target is effected.
Excellent suggestions by everybody here. In general I agree that if you want to demonstrate an interaction between miRNA and gene target, you might want to consider two additional aspects of the work: 1) I'd recommend that before conducting the real experiment you need to optimize the concentration of both vectors. Usually, transfection experiments always use much higher DNA concentrations, which can lead to interaction between these molecules, when in fact under natural condition these may not interact at all; in my lab we usually titrate both vectors and use the lowest conc. where the signal is still robustly detected. 2) another thing to remember, these are artificial systems; if you use luc assays, you are cloning only portion of the sequence and even though the miRNA can interact with that sequence in a living cell the entire 3'UTR sequence of the target gene may obscure the target sequence. Again we usually test that by, what I think Dror suggested, transfecting just with the miRNA vector and see if we get reduction of the endogenously expressed target gene. That leads me to my final suggestion, to check if both miRNA and target genes are expressed in NS-1 cell. For that you don't need any transfection so it should be pretty straightforward. If both molecules are expressed, assuming that all other experiments are positive, you'd have a pretty strong argument that your miRNA and target gene will interact in these cells. Just a thought. Good luck
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